The long-term goal of our studies is to determine the mechanisms by which human papillomaviruses regulate productive replication in differentiating epithelial cells. During productive infection, HPV genomes are stably maintained as low copy episomes in undifferentiated basal cells, while genome amplification and virion assembly occur in highly differentiated suprabasal cells. Our recent work has demonstrated that HPV proteins activate the ATM DNA damage response and that this is necessary for genome amplification in differentiating cells. Interestingly only a subset of ATM pathway members are activated by HPV proteins upon differentiation. Further studies indicate that E7 binds to ATM and this may be responsible for activating a subset of ATM pathway members. We have also demonstrated that HPV proteins also induce low levels of caspase cleavage upon differentiation and that this is also necessary for productive replication in differentiating cells. The ATM pathway appears to be linked to caspase activation as inhibitors of Chk2 block caspase cleavage. These observations form the basis of the proposed studies to examine the role that DNA damage pathways play in HPV amplification in differentiating cells. In this application we will ask the following questions: 1: Which members of the ATM pathway play essential roles for productive viral replication in differentiating cells? Why is ATM pathway important for amplification in differentiating cells? 2). How does E7 contribute to activation of the ATM pathway? Could other HPV proteins play a role? 3). What role does the ATM pathway play in differentiation-induced activation of caspases? How does E7 activate this pathway? PUBLIC HEALTH RELEVANCE: Cervical cancer is the second leading cause of death by cancer in women worldwide and the overall 5-year survival rate is approximately 50%. In 2006, the FDA approved a prophylactic Human Papillomavirus (HPV) vaccine but this vaccine will not protect women against all the HPV types that cause cervical cancer. In addition, it is ineffective in clearing existing lesions as it only blocks initial infection. This study examines how the HPV replication is modulated through the action of the ATM DNA damage pathway. This is an area of high importance as it can identify new targets for anti-viral treatments.